1. Field of the Invention
The present invention relates to a refolding method for restoring the native higher-order structure of a protein which has lost activity and/or stability as a result of becoming insoluble or losing its higher-order structure.
2. Brief Description of the Related Art
When preparing a recombinant protein using a production host, such as E. coli, the protein can become denatured and/or insoluble in water, also known as denatured. Often, the protein has also lost its activity and/or become destabilized. Therefore, the higher-ordered structure of its native state must be restored if the protein is to be used in a pharmaceutical preparation or the like. Protein refolding methods can be used in such cases.
However, reagents and methods used in refolding must be appropriately selected for each protein; therefore, such techniques can be difficult for a person with no experience in refolding. In addition, even for a person with sufficient experience, it still may be difficult to re-acquire the native state of a protein if the protein has a complicated higher-ordered structure and is likely to associate and/or aggregate during refolding.
In order to resolve these problems, several novel refolding methods have been proposed. For example, the following methods and combination methods or modified methods thereof have been reported: a column-refolding method in which the risk of association and/or aggregation is lowered by using a chromatography column (A. Jungbauer, W. Kaar, R. Schlegl: Current opinion in Biotechnology 15, 487-494 (2004)); an immobilized refolding method in which a target is bound to a carrier to prevent its association and/or aggregation (M Matsubara, et al.: FEBS LETT., 342, 193-196 (1994)); a pH extraction method in which a protein is solubilized in a partially-denatured state using an acidic or basic buffer instead of a denaturing agent (S. M. Singh and A. K. Panda: Journal of Bioscience and Bioengineering 99, 303-310 (2005)); a method in which a molecular chaperone is used in combination (D. Rozeman and S. H. Gellman: Journal of Biological Chemistry 271, 3478-3487 (1996)); a method in which an reversed micelle is used to exert a molecular chaperone-like function (A. J. Hagen, T. A. Hatton, and D. I. C. Wang: Biotechnol Bioeng. 35, 955-965 (1990)); a method in which a micelle of a surfactant is utilized (G. Zardeneta and P. H. Horowitz: Analytical Biochemistry 223, 1-6 (1994)); and a high-pressure refolding method in which extraction is carried out under an ultra-high pressure exceeding 3000 atmospheres without using any denaturing agent (M. B. Seefeldt, Y. S. Kim, J. Carpenter, T. W. Randolph: Protein Science 14, 2258-2266 (2005)).
The technique which has attracted the most attention among these is the “artificial chaperone system”, which is a multi-stage refolding method. In this method, the objective protein is extracted with a denaturing agent and then diluted with a buffer containing a surfactant to recover a partial structure while preventing association and/or aggregation. In the next step, the surfactant is forcibly stripped from the protein with a surfactant binder, such as cyclodextrin, and the re-folded higher-order structure is formed. The advantage of this technique has been considered that it is possible to effectively prevent association and/or aggregation, which is the most serious problem, without carrying out trial-and-error experiments, simply by searching several conditions according to a predetermined method. A successful case in refolding by using this technique has been reported, and a modified method of this method has been continuously studied (S. Machida, S. Ogawa, S. Xiaohua, T. Takaha, K. Fujii, K. Hayashi: FEBS Lett. 486, 131-135 (2000)).
However, as the artificial chaperone system was increasingly used, the following facts and the like were revealed: the stripping of the added surfactant from the protein is not as easy as reported; and complicated experiments are still required in order to determine the appropriate re-folding conditions. In addition, the multi-stage operation complicates the process, and therefore limits the application to industrial-scale production (H. Lanckriet and A. P. J. Middelberg: Biotechnology Progress 20, 1861-1867 (2004)).
As described above, even the artificial chaperone system which has received the highest evaluation is not sufficient as a method which allows a person with no experience to easily and effectively carry out protein refolding.
Alternatively, a method has been reported for refolding a protein using acylated sarcosine. Acylated sarcosine cannot be stripped from a protein when simply diluted; therefore, the native higher-order structure of the protein cannot be restored unless the acylated sarcosine is removed by a special method (Richard Burgess: Methods in Enzymology. 273, 145-149 (1996), and EP 0263902 A). A method has also been reported of refolding in which insoluble bovine growth hormone is solubilized by a surfactant solution containing lauroyl-L-glutamic acid, and making the solution strongly alkaline, and then the concentration of the surfactant is lowered by ultrafiltration (U.S. Pat. No. 6,410,694). In this method, it is difficult to effectively restore the native state of any protein other than the growth hormone. In addition, when using a strong alkaline pH environment, the protein can be chemically changed, such as deamidation, which is irreversible.